Osteoporosis is an extremely common degenerative disease resulting from an imbalance between bone formation and bone resorption. This discrepancy worsens with age, such that approximately 50% of women over the age of 50 will experience an osteoporotic fracture. The molecular mechanisms guiding the differentiation of bone forming cells, or osteoblasts, are not clearly understood. Gsa is a ubiquitously expressed G protein subunit that mediates signaling cascades downstream of a variety of G protein-coupled receptors (GPCRs). Several GPCRs have been implicated in skeletal development, and in particular the parathyroid hormone (PTH)/PTH-related peptide receptor (PPR) has a critical function in osteoblast development. We have hypothesized that Gsa likely has essential functions at multiple stages of bone development and that ablation of Gsa early in the osteoblastic lineage will have profound effects on osteoblast differentiation. We have conditionally deleted Gsa in early osteoblast precursors by mating Gsafloxed mice with transgenic mice carrying the Cre recombinase driven by the promoter of osterix, a transcription factor expressed early in osteoblastogenesis. Mice with deletion of Gsa signaling early in osteoblast differentiation have marked skeletal fragility as demonstrated by numerous postnatal fractures and severely reduced trabecular and cortical bone. Furthermore expression of osteocalcin, a marker of terminally differentiated osteoblasts, is almost absent. TRAP staining for osteoclasts does not reveal dramatic increase in bone resorption, making a failure of bone formation the more likely etiology for reduced bone mass in these mutant mice. The focus of this grant proposal will be on the impact of Gsa deficiency on osteoblast differentiation. The expression of green fluorescent protein (GFP) regulated by osterix in these genetically altered mice further provides the opportunity to determine how and whether commonly used in vitro assays for osteoblast differentiation correspond to actual events in vivo. Aim I seeks to determine how Gsa regulates osteoblast differentiation; this will be assessed in vivo using in situ hybridization, and in vitro by culturing bone marrow stromal cells from wild-type and BGsaKO mice under osteogenic conditions. Furthermore, flow cytbmetry and fluorescence-activated cell sorting (FACS) will be applied to isolate cells of the osteoblast lineage in order to determine the mechanisms used by Gsa to direct osteoblast differentiation. Bone formation is dramatically stimulated by both PTH and canonical Wnt signaling pathways, and Aim 2 seeks to determine whether the loss of Gsa-mediated signaling results in inhibition of Wnt signaling as a mechanism underlying the profoundly reduced bone mass in BGsaKO mice. Finally, Aim 3 will focus on determining the ability of osteoprogenitors to differentiate towards other mesenchymal lineages such as adipocytes, using lineage tracing in vivo and cell differentiation assays in vitro.